Glutamine synthetase (GS) is a critical enzyme in the synthesis of the amino acid L-glutamine. See, Meister, A. in Glutamine Metabolism, Enzymology and Regulation (eds. J. Mora & R. Palacios) 1-40 (Academic Press, N.Y.; 1980). A GS-negative cell line is therefore auxotrophic for L-glutamine. GS is frequently used as a selection marker gene in CHO cell based recombinant protein expression systems (Wurm et al. (2004) Nature Biotechnology 22: 1393-1398), though the absence of a GS-negative CHO line requires the use of the GS inhibitor methionine sulfoximine to achieve selection.
In addition, dihydrofolate reductase (DHFR, 5,6,7,8-tetrahydrofolate:NADP+oxidoreductase) is an essential enzyme in both eukaryotes and prokaryotes and catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential carrier of one-carbon units in the biosynthesis of thymidylate, purine nucleotides, glycine and methyl compounds.
DHFR-deficient cells have long been used for production of recombinant proteins. DHFR-deficient cells will only grow in medium supplemented by certain factors involved in folate metabolism or if DHFR is provided to the cell, for example as a transgene. Cells into which a DHFR transgene has been stably integrated can be selected for by growing the cells in unsupplemented medium. Moreover, exogenous sequences are typically co-integrated when introduced into a cell using a single polynucleotide. Accordingly, when the DHFR transgene also includes a sequence encoding a protein of interest, selected cells will express both DHFR and the protein of interest. Furthermore, in response to inhibitors such as methotrexate (MTX), the DHFR gene copy number can be amplified. Accordingly, sequences encoding a protein of interest that are co-integrated with exogenous DHFR can be amplified by gradually exposing the cells to increasing concentrations of methotrexate, resulting in overexpression of the recombinant protein of interest. However, despite the wide use of DHFR-deficient cell systems for recombinant protein expression, currently available DHFR-deficient cell lines do not grow as well as the parental DHFR-competent cells from which they are derived.
Thus, mammalian cells with single and multi-gene knockouts have enormous utility in research, drug discovery, and cell-based therapeutics. However, conventional methods for the targeted elimination of an investigator-specified gene rely upon the process of homologous recombination or gene targeting. Mansour et al. (1988) Nature 336:348-352; Vasquez et al. (2001) Proc Natl Acad Sci USA 98:8403-8410; Rago et al. (2007) Nature Protocols 2:2734-2746; Kohli et al. (2004) Nucleic Acids Research 32, e3. While capable of generating a defined biallelic knockout, for many cell types this technique has proven too inefficient and thus too laborious for routine application. See, e.g, Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-622. These methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Consequently, the generation of mammalian cell lines modified at multiple target loci has been largely unexplored.
Zinc-finger nucleases (ZFNs) have been used for targeted cleavage and gene inactivation. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; 2008/0015164 and U.S. Ser. No. 12/218,035 and International Publication WO 07/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. Formed via the fusion of an engineered zinc-finger DNA binding domain specific for a designated target sequence and the catalytic domain of Fok I (a restriction endonuclease from Flavobacterium okeanokoites), ZFNs provide the ability to place a double-strand DNA break (DSB) at a chosen genomic address. The removal of this site-specific DSB is carried out by the cell's own DNA repair machinery either via a homology-directed repair process when donor DNA is provided, or via non-homologous end joining (NHEJ). See, e.g., Urnov et al. (2005) Nature 435:646-651 (2005); Moehle et al. (2007) Proc Natl Acad Sci USA 104:3055-3060 (2007); Bibikova, et al. (2001) Mol Cell Biol 21:289-297; Bibikova et al. (2003) Science 300:764; Porteus et al. (2005) Nature Biotechnology 23:967-973; Lombardo et al. (2007) Nature Biotechnology 25:1298-1306; Perez et al. (2008) Nature Biotechnology 26:808-816; Bibikova et al. (2002) Genetics 161:1169-1175; Lloyd et al. (2005) Proc Natl Acad Sci USA 102:2232-2237; Morton et al. (2006) Proc Natl Acad Sci USA 103:16370-16375.
While both homology-directed repair and NHEJ processes result in the modification of the target locus, the NHEJ-driven approach obviates the need for donor DNA design and synthesis yet results in a high frequency of disrupted alleles and the error-prone nature of the NHEJ-mediated DSB repair can be exploited to achieve the knockout of a targeted gene in mammalian cells following simple transient transfection of a DNA construct encoding the ZFNs. See, e.g., Santiago et al. (2008) Proc Natl Acad Sci USA 105:5809-5814. ZFN technology has allowed the isolation of several independent knockout cell lines from a screening effort of less than one 96-well plate of single-cell derived clones. As no donor DNA or selection strategy was employed, the resultant single-gene knock out line is a suitable starting cell line for subsequent genetic modification.